PROJECT SUMMARY Post-translation modification (PTM) of chromatin, consisting of DNA and histone proteins, allows for dynamic signaling processes necessary to maintain cellular phenotypes. Mutations in the protein machinery that writes, reads, and erases chromatin marks are frequently present in cancers and, in many cases, even drive oncogenesis. Only recently have mutations in histones themselves been linked to cancers. These so-called ?oncohistones,? some of which act in a dominant fashion, occur with high genetic penetrance in cancers such as pediatric gliomas (H3K27M, H3G34R), chondroblastomas (H3.3K36M), and giant cell tumors of bone (H3.3G34W/L). The discovery of these oncohistones led us to speculate whether histone mutations exist in other cancers. To address this question, we mined both publically available and unreported institutional sequencing data from tumor samples, including 61 different cancer types, to generate an oncohistone database of 2,756 histone missense mutations. Among the most abundant of these new histone mutations are N-terminal arginine mutations. Like previously identified oncohistones, these mutations are located near key regulatory PTMs on histone tails (e.g. H3K4 and H3K27) and are themselves sites of modification (e.g. H3R2 and H3R26). In the proposed research, we will test the hypothesis that these histone arginine mutations, either directly (through altered interactions with their cognate writers) or indirectly (via altered PTM crosstalks), contribute to the development and/or progression of cancer. The goals of this research are two-fold: (1) to investigate the cellular consequences of histone N-terminal arginine mutations and (2) to determine their effects on histone PTM deposition. We will evaluate the cellular consequences of these novel oncohistone mutations in a high- throughput manner in ?humanized? yeast strains, assessing growth, nucleosome accessibility, and transcriptional status. We will complement these yeast studies by expressing tagged histone mutants in mammalian cells, performing pull-downs, and assessing changes in PTMs using proteomic techniques. To assess the biochemical consequences of these mutations on histone PTM deposition, we will generate a DNA-barcoded histone mutant nucleosome library and incubate it with relevant PTM writers. In instances of altered PTMs, we will perform follow-up binding and kinetics experiments to characterize these modified interactions. These experiments will provide critical insight into the cellular and biochemical consequences of histone N-terminal arginine mutations in the context of cancer. Moreover, the two experimental platforms developed herein?one for cellular phenotypic screening and one for biochemical PTM alteration screening?will bolster efforts by the broader epigenetics community to understand the expanding role of histone mutations in cancer development and progression.